CN1763005A - Preparation method and medical uses of N(1)-hydrocarbyl-3'-nitrotylindirubin derivative 1 - Google Patents

Abstract

The present invention is preparation process and antitumor effect of N(1)-alkyl-3'-oximino indirubin derivative (I, JN-2528). The new synthesis process uses indirubin as initial material, and under the action of alkaline compound as promoter, indirubin is reacted directly with halohydrocarbon or other halide to obtain N(1)-alkyl indirubin derivative, which is then reacted with hydroxylamine to obtain N(1)-alkyl-3'-oximino indirubin derivative. The MTT and SRB process and transplanting C57 mouse Lewis lung cancer mold test proves the antitumor activity of N(1)-alkyl-3'-oximino indirubin. The compound shows extracorporeal and intracorporeal cancer inhibiting effect.

Description

The preparation method and the medical use thereof of a kind of N (1)-alkyl-3 '-oximido derivatives of indirubin (I)

Technical field

The present invention relates to the Indirubin be starting raw material prepare N (1)-alkyl-3 '-the oximido Indirubin (structural formula I, novel method JN-2528), with and antitumor, the medical applications of diseases such as treatment senile dementia and anti-virus infection.

Background technology

N (1)-alkyl-3 '-oximido derivatives of indirubin (I) is known cell-cycle kinases (CDKs) ^[1-4], Stat3 information conducting system ^[5]With glycogen synthase kinase-3 β (GSK-3 β) ^[6-8]Inhibitor, owing to exist the CDKs overexpression in most of malignant cells or/and the situation of corresponding gene amplification, so suppressing CDKs is the target of a proper treatment cancer and cell proliferation abnormal diseases ^[4,9]Nerve degeneration is too much relevant with the GSK-3 β in the neural system with disorders, thus N (1)-alkyl-3 '-oximido derivatives of indirubin (I) is the treatment nervous system disorders, such as senile dementia etc. ^[10]The potential active drug; For virus as virus of AIDS (HIV) ^[11]And cytomegalovirus ^[12]Deng, this class derivatives of indirubin all has killing action.Therefore, the novel method of synthetic this class derivatives of indirubin of research and the medical applications of compound thereof extremely arrive people's attention.

Existing prepare N (1)-alkyl-3 '-method of oximido derivatives of indirubin (I) mainly contains following two kinds:

Most bibliographical informations prepare N (1)-alkyl-3 '-method of oximido derivatives of indirubin (I), be with isatin (Isatin, or title isatin) be starting raw material, get N (1)-alkyl isatin through hydrocarbonylation, get N (1)-alkyl derivatives of indirubin (II) through condensation with 3-indanol (3-Indolol, or title 3-Indoxyl) again ^[13-16][Eisenbrand.EP 98109845.2,1998-5-29; WO 99/62503,1999-12-9; Eisenbrand.EP 99706207.6,1999-4-12; WO 00/61555,2000-10-19], last, again with azanol reaction, make Compound I (seeing figure-2).

This method reactions steps is more, is three steps, when the second step preparation N (1)-alkyl derivatives of indirubin (II), the normal bisindole derivatives isomer that produces, as indigo etc., and other impurity, bring difficulty to separation and purification, influenced the productive rate of II, this is the defective place of this method.

Prepare N (1)-alkyl derivatives of indirubin (II) with phase transfer catalysis process, this method is raw material with the Indirubin directly, in multiphase medium 25%NaOH-THF, through phase-transfer catalyst TEBA (triethyl benzyl ammonia chloride) effect, makes intermediate II with the RX reaction ^[17,18], then, similar to figure-2, II and azanol reaction make Compound I, see figure-3.

This method reactions steps reduces, but owing to react in strong alkali aqueous solution with among the THF, has destroyed the hydrogen bond between 2-carbonyl and N (1 ')-hydrogen atom, make alkylation reaction lose selectivity, part N (1 ')-position difficult the separation, influences the yield and the purity of Compound I also by hydrocarbonylation.

On the other hand, whether the decision compound has medical applications to be worth and have following prerequisite at least: 1) drug molecule has and target molecule, and target cell or target organ interact and correct the ability of its pathology pathology; 2) drug molecule has the ability that arrives target by microbial film; 3) drug molecule should have preferably selectivity and then less toxic side effect is arranged.

Indirubin is the compound of two indoles heterocycle two dimensional structures, its molecular size by chance with cell-cycle kinases (CDK) in ATP-binding site suitable, and then with cell in ATP strive unexpectedly and combine with CDK and suppress the CDK activity.But because Indirubin can not form the H key with CDK albumen, make that the binding affinity between Indirubin and CDK is little, to the enzymic activity restraining effect a little less than.In The compounds of this invention, we Indirubin 3 '-introduced oximido on the position, can be formed centrally stable hydrogen bond during ATP in the CDK molecule combines, and then strengthened this compound and CDK avidity and had stronger enzyme inhibition.But because two indoles heterocycle two dimensional structures of Indirubin have bigger polarity, this physico-chemical property has determined that it is water-soluble and oil soluble is all very poor, is difficult for arriving the drug effect position by microbial film, and bioavailability is poor.The Compound I ndirubin-3 ' in the Europatent-monoxime-5 Sulphonic acid (IMSA) [EP1079826B1] for example, though in acellular experimental system, very strong CDK restraining effect is arranged, but this compound polarity is very big, can't pass through cytolemma, so we have proved no any biological function in cell system, see experimental example two.

Summary of the invention

The object of the invention is: to provide with the Indirubin be feedstock production N (1)-alkyl-3 '-the easy synthetic method of oximido derivatives of indirubin (I).

The object of the invention also is: make to be reflected under the anhydrous state and carry out, avoid producing isomer or other by product in the process of preparation intermediate II, be convenient to the purifying of intermediate II, help the synthetic and purifying of final product.

The present invention also aims to seek N (1)-alkyl-3 '-application of oximido derivatives of indirubin in treatment and preventing cancer.By in the Indirubin molecule, introducing two important chemical functional groups: 3 '-oximido and N-alkyl, make resulting novel derivative have bigger CDK avidity and bigger fat-soluble, and then strengthened biologic activity and bioavailability and have medical proper value.

Technical scheme of the present invention is: N (1)-alkyl-3 '-oximido derivatives of indirubin (I) preparation method comprises following two-step reaction:

Step 1, Indirubin is dissolved in the anhydrous organic polar solvent, after the cooling, adds basic-type accelerator, then add halohydrocarbon RX in batches, R is C1～5 alkyl, C2～5 alkene and aryl etc. here; X is Br or I.Through reaction, optionally make single N (1)-alkyl derivatives of indirubin (II);

Step 2, intermediate II is added among aqueous pure R ' OH, after the dissolving, add hydroxylamine hydrochloride, add a little alkaline matter, with in and hydrochloric acid, be heated to backflow then, after a few hours, reaction finishes, make N (1)-alkyl-3 '-oximido derivatives of indirubin (I).

The process of above-mentioned preparation N (1)-alkyl-3 '-oximido derivatives of indirubin (I) can be represented with following equation:

In the technique scheme, the anhydrous polar solvent that uses in " step 1 " is tetrahydrofuran (THF) (THF) or N, dinethylformamide (DMF) or N,N-dimethylacetamide (DMA) etc., and outstanding two kinds of solvents later on are good; Cooling temperature is between-10-5 ℃; Added basic-type accelerator comprises inorganic base substance, as Na ₂CO ₃, K ₂CO ₃, NaOH, KOH and NaH etc., and strong organic basic material, as DBU etc., the mol ratio of they and starting raw material Indirubin is 1.0～1.8; More preferably basic-type accelerator is NaH and DBU, and the mol ratio of they and raw material Indirubin elects 1.3～1.6 as, and reinforced and temperature of reaction are-2～5 ℃.

In the technique scheme, the halohydrocarbon RX that uses in " step 1 ", R is C1～5 alkyl, C2～5 alkene and aryl etc.; When R is alkyl, can be straight chain, branched and corresponding cyclic alkyl; When R is alkene, be generally monoolefine, can be straight chain or band branched-chain alkene; When R is aryl, often select benzyl for use, have the phenyl of single or multiple electron-withdrawing groups.X is Br or I (iodine).The mol ratio of RX and starting raw material Indirubin is 1.0～1.8, more preferably 1.3～1.6.

In the technique scheme, reaction finishes to be determined as foundation with TLC in " step 1 ", after the end, solvent can be removed under the cryogenic vacuum condition, be poured in the frozen water under perhaps directly reaction solution being stirred, separate out intermediate II, after filtration, washed several times with water gets crude product II, through methods such as recrystallization or purification by silica gel column chromatography, get high N (the 1)-alkyl derivatives of indirubin (II) of purity.The II that makes through above-mentioned technical qualification is single N (1)-alkyl Indirubin, its structural identification see N among the embodiment (1)-alkyl-3 '-oximido derivatives of indirubin (I) crystal X-diffraction structure qualification result.

In the technique scheme, in " step 2 " by intermediate II and azanol reaction make N (1)-alkyl-3 '-oximido derivatives of indirubin (I), its preparation is to be solvent with pure R ' OH, and the R ' here is methyl, ethyl, propyl group, sec.-propyl etc., is excellent to select preceding two kinds of alcohol for use; Water content is good with 15～25% between 5～35% in the alcohol; After adding intermediate II, the alkaline matter of interpolation is NaOH or KOH or sodium alkoxide and potassium alcoholate etc., and its consumption reaches pH value＞6 that make reaction solution and is standard, and then, reflux detects the demonstration reaction until TLC and finishes.The alkali number of return time and adding is inverse ratio.

In the technique scheme, after reaction finishes in " step 2 ", the solvent vacuum concentration is removed, added entry; Or directly reaction solution is poured in the big water gaging; With after the acid neutralization, separate out N (1)-alkyl-3 '-oximido derivatives of indirubin (I) crude product solid, behind the filtration washing, drying gets the pure product of Compound I through recrystallization.Recrystallization carries out in polar solvent, and with acetone or acetonitrile, or their mixed solution is good.

The invention has the beneficial effects as follows: be raw material with the Indirubin, with other raw and auxiliary material that is easy to get carry out fairly large production N (1)-alkyl-3 '-oximido derivatives of indirubin (I), its method economy, simple and direct.

Among the present invention, the intermediate II that the first step reaction is produced is single, selected reaction conditions has avoided isomer or other by product to produce and accumulation, is easy to the intermediate II purifying, thus form a kind of preparation high purity N (1)-alkyl-3 '-novel method of oximido derivatives of indirubin (I).

Above-mentioned N (1)-alkyl-3 '-oximido derivatives of indirubin (I) preparation method is Indirubin to be dissolved in N, dinethylformamide (DMF) or N, in the N-N,N-DIMETHYLACETAMIDE (DMA), under-2～5 ℃ of conditions, add NaH or DBU, the mol ratio of they and raw material Indirubin elects 1.3～1.6 as; Then, add halohydrocarbon RX, the mol ratio of RX and starting raw material Indirubin is 1.3～1.6.Reaction finishes to be determined as foundation with TLC, after the end, will pour in the frozen water under the reaction solution stirring, separate out intermediate II, after filtration, washed several times with water, get crude product II,, get high N (the 1)-alkyl derivatives of indirubin (II) of purity through methods such as recrystallization or purification by silica gel column chromatography.Intermediate II and azanol are to reflux in 15～25% methyl alcohol or the ethanol in water content, make the Compound I crude product, through acetone or acetonitrile recrystallization, N (1)-alkyl-3 '-oximido derivatives of indirubin (I).

More excellent N (1)-alkyl-3 '-oximido derivatives of indirubin (I) preparation method is, Indirubin is dissolved in N, in the dinethylformamide (DMF), under-2～5 ℃ of conditions, adds NaH, and the mol ratio of it and raw material Indirubin elects 1.3～1.5 as; Then, add halohydrocarbon RX, the mol ratio of RX and starting raw material Indirubin is 1.3～1.5.Reaction finishes to be determined as foundation with TLC, after the end, with pouring in the frozen water under the reaction solution stirring, separates out intermediate II, and after filtration, washed several times with water gets crude product II, through purification by silica gel column chromatography, gets high N (the 1)-alkyl derivatives of indirubin (II) of purity.Intermediate II and azanol are to reflux in 25% ethanol in water content, make the Compound I crude product, through acetone recrystallization, N (1)-alkyl-3 '-oximido derivatives of indirubin (I).

N (1)-alkyl-3 '-oximido derivatives of indirubin (I) is the CDK inhibitor, can be used for tumor treatment and prevention.Its structural formula is as follows:

The tumour of indication comprises various leukemia and various solid tumor.

In use, can be single therapy, can also be combination therapy.Combination therapy can be and other chemotherapy coupling, can also with the herbal medicine coupling, or with operation, radiotherapy, immunotherapy, hormonotherapy, couplings such as gene therapy.Combination therapy can be to treat simultaneously, can also be different precedence treatments.

The JN-2528 series compound can adopt any medicine-feeding way as medicinal, any administering mode.Promptly can be oral, injection sucks, and Transdermal absorption is implanted, cavity, mucous membrane, and intravenous drip etc.

The JN-2528 series compound can be made tablet as medicinal, pill, and capsule, paste, creme, patch, pulvis, injection, sprays, implant is fastened agent, multiple formulation such as drops.

The present invention shows Anticancer Activities by MTT and srb assay, and NJ-2528 is to mammary cancer, liver cancer, lung cancer, cancer of the stomach, colorectal carcinoma, cervical cancer, melanoma, hormone-dependent type and hormone independent form prostate cancer and neuroblastoma all have the good anticancer effect.Particularly importantly JN-2528 also has the restraining effect identical with hormone-dependent prostate cancer to the hormone independent form prostate cancer of feeling simply helpless clinically.

Being noted that JN-2528 in tumor therapeutic procedure, can be single therapy, can also be and 2 kinds 3 kinds or more multiple medicines thing combination therapy, administration simultaneously, or different precedence administration.Here said combination therapy can be and other Western medicine that herbal medicine merges use, or and hormonotherapy, radiotherapy, immunotherapy, chemotherapy, cold therapy and gene therapy combined treatment.

The medicament production treatment tumour that this invention provides, these products contain the JN-2528 of effective dose and all suitable additives that other pharmaceutical industry is adopted, and various suitable medicine-feeding way.Consummate drug manufacture technology comprises that the prodrug that uses nontoxic pharmaceutical industry to accept is raw material, adopts several different methods to extract from natural materials or synthetic JN-2528.Consummate drug manufacture technology also comprises uses solvent that pharmaceutical industry the accepts solvent as JN-2528, water for example, and mineral oil, edible oil, dimethyl sulfoxide (DMSO), etc.

JN-2528 can be made into oral administration, local application, inhalation, formulations such as anum administration.Use material that nontoxic pharmaceutical industry accepts as carrier, additives, excipient.Be pointed out that once more administering mode can be a drug combination.Oral administered dosage form comprises: tablet, capsule, tincture, lozenge, oral syrup or other all suitable formulations.

JN-2528 can be made into the formulation that is administered systemically, and comprising: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraspinal injection, intrathecal injection, or other any drug administration by injection and instillation.In addition, the JN-2528 preparation still can contain other pharmaceutically all available non-toxic substances except that the JN-2528 that contains effective dose.Comprise one or more carriers, thinner, additives, and if necessary, other medicinal component.

JN-2528 can be made into any type of oral dosage form, comprising: tablet, and lozenge, the soft or hard capsule can be obeyed aqua, tincture, oral administration mixed suspension, pulvis, emulsifying agent etc.Oral dosage form can contain one or more sweeting agents, correctives, and color agent and sanitas are to guarantee that color is perfect and to be convenient to take.

The excipient that is used for the JN-2528 tablet can be the inert thinner, as lime carbonate, and yellow soda ash, calcium lactate, calcium phosphate, sodium phosphate; System granule: W-Gum, alginic acid; Tackiness agent: starch, gelatin or gum arabic; Slipping agent: stearic acid, Magnesium Stearate or talcum powder.Adopt all known technologies, make dressing or the tablet of having no clothes.Coated dosage form comprises enteric coating and slow release formulation.The material that is used for slow release formulation can be glycerine monoester or dialycerides salt.

The JN-2528 capsule can be a hard capsule, can also be soft capsule.The auxiliary material that is used for hard capsule can be lime carbonate, calcium phosphate or kaolin.Be used for the auxiliary material such as the water of soft capsule, oils comprises peanut oil, sweet oil or liquid paraffin.

The oral water type of JN-2528 suspension can be formulated with the JN-2528 and the suitable diluent of effective dose.Its dilution suspensoid can be sodium cellulose glycolate or methylcellulose gum, Vltra tears, alginic acid sodium salt, polyvinylpyrrolidone, tragcanth, gum arabic.Dispersion agent or wetting agent comprise: natural phospholipid, as Yelkin TTS, or the condenses of alkene oxide and lipid acid, as 17 alkyl oxidation of ethylene alkanols, or ethene and fatty acid part esterification condensation thing, or hexitol such as the single oleate of polyoxyethylene sorbitol, or ethylene oxide and fatty acid part fat condensation product, and alcohol anhydride, as polyethylene sorbyl alcohol methyl esters hydrochlorate.

The oral water type of JN-2528 suspension also may contain one or more sanitass, as ethyl to or o-hydroxy st-yrax salt; Contain one or more sweeting agents, as sucrose or asccharin.

JN-2528 oil type suspension can be suspended in the JN-2528 of effective dose in the vegetables oil, as soybean oil, and peanut oil, Semen Maydis oil, sweet oil, sesame oil, theobroma oil.Or mineral oil, as liquid paraffin.Said preparation also can contain thinner, as beeswax, and wax, or cetyl alcohol, sweeting agent mentioned above, and correctives and stablizer are as antioxidant vitamin C (xitix).

Adopt aforesaid dispersion agent, wetting agent, suspensoid, sanitas, correctives, sweeting agent, and tinting material, JN-2528 also can be made into oil-in-water preparation.JN-2528 makes oil-in-water emulsifiers.Its oil phase can be aforesaid vegetables oil, as soybean oil, and sweet oil, peanut wet goods.Can also be liquid paraffin, mineral oil, or the mixture of two classes.Emulsifying agent can be natural emulsifying agent, as kordofan gum, or tragcanth, natural phospholipid such as soybean phospholipid, Yelkin TTS, lipid acid, the fat of hexitol or acid anhydride or half resin are as the sorbose monoester.Also artificial chemical materials, as ethylene oxide half fat condenses, polyethylene sorbyl alcohol monoester salt.The oil-in-water type emulsion preparation of JN-2528 also can contain sweeting agent and correctives etc.

The syrup of JN-2528 and tincture can adopt sweeting agent, as propyl alcohol, and propylene glycol, sorbyl alcohol or sucrose.Said preparation also can contain wetting agent, sanitas, correctives, color additives.

The injection of JN-2528 can adopt aseptic parenteral solution or oily suspension.It can use all dispersion agent, wetting agent or suspensoids that are suitable on the pharmaceutics.Said preparation can be aseptic transparent liquid, uses all thinners that are suitable on the pharmaceutics, as 1,3 butylene glycol, and water, Ringer's solution, or physiological saline.In addition, aseptic lipid also can be used as solvent or suspensoid.It can be synthetic glycerine monoester or two fat, and lipid acid is as oleic acid etc.

JN-2528 also can be made into suppository, as supp anal etc.Suppository can be combined by a certain amount of JN-2528 and corresponding non-stimulated excipient.Excipient is solid at normal temperatures, but liquefies and the release medicine under anus body temperature.The excipient that is suitable for comprises theobroma oil and macrogol class.

The formulation that is administered systemically of JN-2528 according to used carrier concentration, can be a suspension type, also lysotype.For easy-to-use or alleviate patient's misery, can add sanitas, buffer reagent and local anesthetic.

JN-2528 also can be used as animal doctor's medication.Take for ease of animal, the preparation that contains JN-2528 can add in the animal-feed or in the drinking-water.Also can be made into easily formulation as food or the drink of animal.

For the people, during above-mentioned disease, the dosage range of JN-2528 tentatively is decided to be per kilogram of body weight 0.01mg to 20mg in treatment, or by 60 kilograms of body weight per capita about every day of 0.6mg to 1200mg.According to administering mode, JN-2528 can be made into single dose formulation or multiple agent type.Dose unit tentatively is decided to be and contains effective ingredient 1mg to 500mg.

Every day, administration number of times was decided according to sick the kind.In most cases, no more than four times of every day.Be to be noted that dosage may depend on multiple factor, as the age, body weight, healthy state, sex, diet, and administration time, approach, the state of an illness and medicine unite use etc.So final dosage regimen should be determined as the case may be by the doctor.

The priority compounds that this patent is mentioned has ideal pharmacology as superior oral administration biaavailability, hypotoxicity, low plasma protein binding ratio and ideal inside and outside transformation period.This compounds can pass through hemato encephalic barrier, and this is an advantage to the treatment central nervous system disease.

The present invention 3 '-N (the 1)-position of oximido Indirubin molecule introduced ethyl, obtain N (1)-alkyl-3 '-oximido Indirubin (JN-2528).Our experiment shows, this patent compound N (1)-alkyl-3 '-the oximido Indirubin is compared with similar compound has following characteristics:

1, in acellular experimental system, JN-2528 is similar to Indirubin-3 '-monoxime-5 Sulphonic acid (IMSA), has stronger CDK restraining effect (IC50 is 0.78 μ M).But IMSA is because of can't passing through the cell biological film, and then this patent has proved that this compound does not have any growth-inhibiting effect to the examination cell.In contrast, no matter JN-2528 is external, or has stronger biologic activity in the body.

2, compare with Indirubin, in acellular experimental system, JN-2528 is to the restraining effect of CDK than Indirubin powerful about 20 times, and simultaneously because the fat-soluble increase of JN-2528, biological activity also obviously is better than Indirubin in the body.

3, traditional tumour chemotherapeutics lacks selectivity to normal cell, so toxicity is big.JN-2528 is the unusual CDK in the anticancer optionally, so normal cell is not had influence, toxic action is extremely low.Under effective antitumor dosage, without any side effects.

Description of drawings

Accompanying drawing 1 be N (1)-alkyl-3 '-structure of oximido derivatives of indirubin (I).

Accompanying drawing 2 is for preparing the most frequently used method of Compound I.

Accompanying drawing 3 prepares Compound I for phase transfer catalysis process.

Accompanying drawing 4 prepares Compound I for patented method of the present invention.

Accompanying drawing 5 is 1-ethyl-3-oxime-Indirubin crystalline structure X-diffraction measurement result.

Embodiment

Experimental example one, the preparation of N (1)-alkyl-3 '-oximido Indirubin:

Step a, 1-ethyl-Indirubin

[step a]: in the solution of being formed by 2.62 gram Indirubins (10mmol) and 40mL dry DMF, add 0.6 gram NaH (15mmol), at N ₂Environment stirs down, and mixture is cooled to 0 ℃, slowly drips and forms solution by 1.15mL iodoethane (15mmol) and DMF (5mL), slowly returns room temperature after adding, and stirs 3 hours, and TLC detects (developping agent is the same).After reaction is finished, stir down and carefully pour in the 300mL frozen water,, merge organic phase, use anhydrous Na with chloroform extraction 3 times (150mL * 3) ₂SO ₄Dry final vacuum is concentrated into dried, and residue gets 1.5 gram purple powder solid 1-ethyl-Indirubins, yield 52% with purification by silica gel column chromatography (elutriant is: chloroform/sherwood oil, 5/1 (v/v)); Fusing point is greater than 200 ℃ (decomposition). ¹HNMR(CDCl ₃)δ：1.33(t，J＝7.2Hz，3H，CH ₃)，3.89(q，J＝7.2Hz，2H，CH ₂)，6.89(m，1H，H-Ph)，6.96(m，1H，H-Ph)，7.00(m，1H，H-Ph)，7.13(m，1H，H-Ph)，7.28(m，1H，H-Ph)，7.52(d，J＝7.6Hz，1H，H-Ph)，7.74(d，J＝7.6Hz，1H，H-Ph)，8.90(d，J＝7.6Hz，1H，H-Ph)，10.56(bs，1H，NH-1)；Anal.Calcd?for?C18H14N2O2(Mr＝290.32)：C?74.47，H?4.86，N?9.65；foundC?74.33，H?4.85，N?9.21.

[step b]: feed intake and reaction process and last consistent, reaction is poured in the 300mL frozen water after finishing, there are a large amount of red-purple solids to separate out (annotating:, be different from method A herein) after the stirring, filter here without chloroform extraction, be washed to neutrality, after the drying, carry out column chromatography purification by method A.

Step b, 1-ethyl-3-oxime-Indirubin

[step a]: 1.2 gram 1-ethyl-Indirubins (2.9mmol) are added in the ethanol of 50mL 75%, then add oxammonium hydrochloride 2.0 grams (29mmol), add KOH in batches, make reaction solution pH transfer to 5～6, reflux 15 hours, and TLC detects (developping agent is the same).After reaction is finished, pour in acidity (a little HCl) water, analyse dark orange red powder solid, after filtration, be washed to neutrality, drying and recrystallization can get 1-ethyl-3-oxime-Indirubin 0.65 gram, yield 75%.1H?NMR(CD3COCD3)δ：1.28(t，J＝7.2Hz，3H，CH3)，3.94(q，J＝7.2Hz，2H，CH2)，6.98(m，1H，H-Ph)，7.07(m，2H，H-Ph)，7.10(m，1H，H-Ph)，7.20(m，1H，H-Ph)，7.35(m，1H，H-Ph)，7.45(m，1H，H-Ph)，8.38(d，J＝7.8Hz，1H，H-Ph)，8.71(d，J＝7.8Hz，1H，H-Ph)，10.56(bs，1H，NH-1)；ESI-MS(70V)m/z：304.1(M-H)-，C18H15N3O2(Mr＝305.3)。

[step b]: 1.2 gram 1-ethyl-Indirubins (2.9mmol) are suspended in the ethanol of 45mL 95%, add 2.0 gram oxammonium hydrochlorides and 8 gram KOH successively, behind the backflow 2h, be chilled to room temperature, pour into again in the 300mL water, the insolubles elimination adds the acetate neutralization in the filtrate, the red solid precipitation of generation, filter, washing, drying, (yield is higher than step a) to get thick product.Available acetone recrystallization purifying.

1-ethyl-3-oxime-Indirubin crystalline structure X-diffraction measurement result is seen figure-5.

Experimental example two, the extracorporeal anti-tumor function of N (1)-alkyl-3 '-oximido Indirubin:

N (1)-alkyl-3 '-oximido Indirubin and N (1)-ethanoyl-Indirubin hormone are to the restraining effect of dependent form and independent form prostate cancer.

What hereinafter exemplify is JN-2528 (N (1)-alkyl-3 '-oximido Indirubin) compound some examples to oncotherapy.We reiterate at this, though we are example with the treatment tumour only, this does not also mean that these compounds only are used for the treatment of tumour.

(1) materials and methods

Reagent: the JN-2528 compound, the synthetic and purifying by our laboratory, with mass spectrum, nuclear-magnetism, infrared, and the X-diffraction is confirmed structure (seeing example one).Purity is＞98.0%.JN-2528 is redness and garnet crystalline powder.During experiment, with the DMSO wiring solution-forming ,-20 ℃ of preservations.European patent similar compound Indirubin-3 '-monoxime-5 Sulphonic acid (IMSA) [EP1079826B1], vitamin A acid, daunorubicin, taxol purchase the Sigma chemical reagents corporation in the U.S..Other chemical reagent of experiment usefulness except that specifying, is all purchased the Sigma chemical reagents corporation in the U.S..

Cell cultures: human carcinoma cell line, mammary cancer, MCF-7; Colorectal carcinoma, LOVO: prostate cancer, male sex hormone dependent form LNCAP and male sex hormone independent form PC-3, DU 145; Neuroblastoma: the N2A cell is purchased the Collection in American Type Culture.Cell places 37 ℃, 5%CO ₂In the incubator, to contain the RPMI1640 culture medium culturing of 10% foetal calf serum and penicillin and Streptomycin sulphate.

MTT and SRB:MTT and SRB experimental technique are as described in the document ^[19], be summarized as follows: the cancer cells in the vegetative period of taking the logarithm, be inoculated in the 96-orifice plate, cell density is every hole 5000 cells/200L.Adding waits the medicine of serial dilution after 24 hours.Cell continues to cultivate 72 hours under drug effect.After the nutrient solution of 100 μ L is taken out in every hole, add 50lMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution continues to cultivate 4 hours.Dissolve the latent throw out of atropurpureus first with 200L hydrochloric acid-aqueous isopropanol, measure absorbance value in 540nm wavelength place.If adopt srb assay, then after 72 hours, remove all nutrient solutions, after cell is fixed 1 hour with 10% Tricholroacetic Acid, put in the air seasoning 24 hours, add 50l SRB (sulforhodamine B) dyeing 20-30 minute, with 1% Glacial acetic acid washing 5 times, put in the air after the seasoning, add 10mM Tris-HCl (pH 10.0) solution dissolving red-purple albumen-SRB mixture of 200l, measure absorbance value in 600nm wavelength place.Try to achieve the mean value of respectively organizing testing data, with experimental group control group is calculated cell and generate inhibiting rate.With Sigma Plot mapping software paint cell growth rate-drug level semilog plot, and try to achieve the medium effective concentration (IC50) of medicine.

(2) result:

Adopt MTT and two kinds of analytical procedures of SRB, we study the antitumour activity of JN-2528.List table 1～3 is JN-2528 and other chemotherapeutics half effective inhibition concentration to various cancer cells.By table 1～3 as can be seen, no matter be mammary cancer (MCF-7, table 1), colorectal carcinoma (LOVO, table 2), hormone-dependent type (LNCAP) and hormone independent form prostate cancer (PC-3, DU-145, table 3), JN-2528 is had certain susceptibility, its half effective inhibition concentration IC50 (sees Table 1-3) between 4～14M, similar to clinical common chemotherapeutics.The result shows that JN-2528 has antitumour activity.

Table 1:JN2528 is to breast cancer cell half effective inhibition concentration (IC50, μ M)

Reach comparison (mtt assay) with other chemotherapeutics.

Medicine	Cell line mcf-7
		JN-2528	6.72±0.54
Daunorubicin	0.054±0.011

Vitamin A acid	21.45±3.78
		NS389	＞200

Table 2:JN-2528 to the half of colon cancer cell suppress effective concentration (IC50, M)

Reach comparison (mtt assay) with other chemotherapeutics.

Medicine	Clone LOVO
		JN-2528	8.49±0.733
Daunorubicin	0.035±0.04
		Vitamin A acid	75.34±12.49
Taxol	0.0037±0.0005
		NS389	＞200

Table 3:JN-2528 to the half of prostate cancer cell suppress effective concentration (IC50, M)

Reach comparison (mtt assay, 3 days) with other chemotherapeutics.

Medicine	Clone
				LNCAP	PC-3	DU145
	JN-2528	10.34±0.42	13.38±0.59				7.44±0.76
Daunorubicin				N/A	0.24±0.02	0.11±0.01
	Vitamin A acid	15.95±3.19	＞50				＞50
NS389				65.54±9.46	＞200	＞200
	Proscar	40.60±7.12	133.68±12.94				N/A
Casodex				57.40±7.21	120.11±17.31	N/A
	Taxol	0.00035±0.0001	0.00296±0.001				0.00577±0.001

(3) discuss

By MTT and SRB experiment, be not difficult to find out that JN-2528 has stronger antitumour activity, its to the cancer cells restraining effect than cell induction differentiation agent vitamin A acid (Retinoid acid, IC50:21.5-＞50M) and new epoxy thing enzyme II inhibitor NS389 (IC50＞200M) much better than.It should be noted that, compare with similar compound Indirubin-3 '-monoxime-5 Sulphonic acid (IMSA) in the Europatent [EP1079826B1], though IMSA cell cycle kinases (CDK) in acellular experimental system has extremely strong restraining effect, we experimental results show that the growth unrestraint effect of IMSA to cancer cells.This is because IMSA polarity is too big, can't pass through microbial film.Most important theories of the present invention according to one of be exactly the physico-chemical property of improving compound, strengthens fat-soluble so that it brings into play pharmaceutical activity by the cell biological film.

JN-2528 also is better than clinical Casodex that uses always (IC50:57.4M) and Proscar (IC50:40.6M) to the restraining effect of hormone-dependent type prostatitis cancer cells LNCAP.What is more important, (PC-3, DU-145), JN-2528 also has the good restraining effect for the hormone independent form prostate cancer cell of feeling simply helpless clinically.By contrast, PC-3 and DU-145 cell have then descended nearly 10 times to taxol susceptibility.

Though compare with daunorubicin or taxol, the JN-2528 antitumous effect is inferior slightly, the former is effectively promptly producing stronger toxic side effect under the drug level, produces serious bone marrow depression when the plasma drug level 0.05M as taxol.And JN-25288 toxicity is very low, and preliminary experiment shows that the mouse LD50 of this compound is approximately 3.5g/kg.

It is worthy of note that though the LNCAP cell is very much responsive to taxol, experiment is difficult to record its IC90, because no matter taxol concentration is much, always the cell of having an appointment about 15-20% is insensitive.Trace it to its cause very likely be these cells under the taxol effect, enter the G0 phase and the further effect of hiding medicine.In contrast, JN-2528 has better curative effect-concentration curve.This result also shows, if JN-2528 is different sequencing administrations with taxol, then may produce apparent synergy.

In addition, JN-2528 does not have significant difference to the IC50 of examination different sorts tumour cell, these are different with other chemotherapeutics, as colorectal carcinoma and breast cancer cell to daunorubicin than the prostate cancer cell sensitivity, this explanation JN-2528 acts on the public target spot that cell generates.

Experimental example three, the anti-tumor in vivo effect of N (1)-alkyl-3 '-oximido Indirubin

(1) materials and methods:

Material: the C57BL/6 mouse, complete male, 25 ± 2g.Nanjing University model animal center provides.The Lewis lung cancer cell strain, Nanjing University of Traditional Chinese Medicine provides.The JN-2528 lyophilized powder, lot number: 050418-1.Every bottle of 1mg adds 1ml physiological saline, is made into 1mg/ml, is provided by Wuxi Jesse pharmaceuticals.Endoxan, Hengrui Medicine Co., Ltd., Jiangsu Prov. produces, lot number 04081921.

Instrument: Sai Duolisi BP-310 electronic balance.Bechtop.Glass homogenizer.The some covers of instruments.

Method: get Lewis lung cancer knurl piece under the aseptic technique, remove necrotic tissue, several knurl pieces are mixed, be cut into small pieces, add PBS and grind, put into centrifuge tube, centrifugal 5 minutes of 1000rpm after mill is even with the glass tissue homogenizer, remove supernatant, add physiological saline and be diluted to 1 in right amount: 3-1: 4 tumor cell suspension.With the suction of 1ml disposable syringe, each suction is preceding with the cell mixing.The right armpit subcutaneous vaccination of mouse 0.2mL tumor cell suspension.Entire operation is finished in 30min as early as possible.

At random be divided into four group with tumor-bearing mice next day, and 10 every group, and the beginning intraperitoneal administration, continuous 10 days.Put to death mouse the next day of last administration, weighs earlier, and the subcutaneous tumors piece is dissected in the back, rejects healthy tissues, claims the knurl net weight.Be calculated as follows inhibiting rate:

Positive controls wherein, every day, an endoxan was annotated in the abdominal cavity, and dosage is the 100mg/kg body weight, continuous 10 days.JN2528 treatment group, dosage is: 15mg/kg and 30mg/kg, the next day abdominal injection once.Non-treatment group (model control group), every day abdominal injection 0.2ml physiological saline.

(2) result and discussion: by experimental result (table 4) as can be seen, though only with 15mg/kg and 30mg/kg dosed administration 5 times, JN-2528 has just shown the effect of tangible anti-Lewis lung cancer, and inhibiting rate is respectively 30.75% and 44.36%.As if compare with the positive drug endoxan, a little less than the JN-2528 effect than endoxan is, but should be noted that endoxan dosage is 100mg/kg, and be administration every day, promptly actual dose approximately is 7 times of JN-2528.Another material facts are that endoxan is a cell toxicity medicament, and is the same with other cytotoxic drug, has effect soon, acts on strong characteristics, but lacks selectivity.Can also find out that from our experiment under used dosage, serious toxic action has appearred in endoxan.It shows with control group and compares, endoxan treated animal weight loss 22%, surpass the weight loss scope (10%) that maximum tolerated dose (MTD) is allowed.In contrast, JN-2528 has good selectivity, under active drug dosage, obviously suppress the generation of Lewis lung cancer, but tangible toxic reaction does not appear in animal.The weight of animals is compared with control group after the medication, does not have any difference.This is consistent with the initial design objective of medicine.Its antitumous effect also can improve by increasing drug dose.Therefore, have reason to believe that JN-2528 has good application prospects.The result shows that JN-2528 group object knurl knurl is heavy to have obvious decline than model group.

Table 4: medicine is to the influence of C57/B6 mouse inoculation Lewis lung cancer (X ± SD)

Group	Number of animals (only)	Dosage (mg/kg)	Knurl heavy (g)	Body weight (g)		Tumour inhibiting rate (%)
							Before the treatment	After the treatment
				Model group	10				0	2.04±0.84	29.5+4.5	37.8+1.7	-
Positive drug	11	100a	0.025±0.008 **			28.40+3.7	21.8+2.3 **	98.79
				JN-2528	12				15b	1.31±0.52 *	25.5+2.54	38.38+2.3	35.76
JN-2528	9	30b	0.91±0.57 **			26.7+1.4	44.38+3.1	55.50

A: administration every day; B: the next day administration; ^*Compare with model control group, ^*P＜0.05, ^*P＜0.01.

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Claims (12)

1, a kind of N (1)-alkyl-3 '-preparation method of oximido derivatives of indirubin (I), comprise following two-step reaction: step (1), Indirubin are dissolved in the anhydrous organic polar solvent, under cooling and basic-type accelerator condition, with halohydrocarbon RX reaction, R is C1～5 alkyl, C2～5 alkene and aryl etc. here; X is Br or I; Optionally make single N (1)-alkyl derivatives of indirubin (II); Step (2), intermediate II are heated to backflow in aqueous pure R ' OH and hydroxylamine hydrochloride under alkaline condition, make N (1)-alkyl-3 '-oximido derivatives of indirubin (I).

2, claim 1 is described with the Indirubin be feedstock production N (1)-alkyl-3 '-reaction equation of oximido derivatives of indirubin (I) is:

3, N (1)-alkyl-3 '-oximido derivatives of indirubin (I) and N (1)-alkyl derivatives of indirubin (II) be as the application of cell cycle kinase inhibitors.

4, cell cycle kinase inhibitors N (1)-alkyl-3 '-oximido derivatives of indirubin (I) and N (1)-alkyl derivatives of indirubin (II) be used for the treatment of malignant tumour in preparation, cardiovascular disorder, uriniferous tubulesization and the ephritis that causes, the neurological disorder disease, psoriasis, and the application in the medicine of AIDS.

5, the application of compound as claimed in claim 4 in preparation treatment disease Chinese traditional medicine, wherein said compound can be made into all suitable formulations in the pharmaceutical industry: little needle injection or middle needle injection or big needle injection or powder needle injection or injectable emulsion or tablet or pill or capsule or paste or creme or patch or liniment or pulvis or sprays or implant or drops or suppository or ointment or confection.

6, the application of compound as claimed in claim 5 in the medicine of preparation treatment disease, wherein said compound adopts various administrations: oral administration, drug administration by injection, drug delivery implant, intracavitary administration, sublingual administration, anum administration, transdermal administration, interior external application.

7, application as claimed in claim 6, wherein said drug administration by injection is: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary administration; Described compound is a single therapy, or unites to use or unite to use or unite to use or unite with biological regulator with surgical operation with herbal medicine with radiotherapy and use or unite use with gene therapy.

8, as claimed in claim 1 prepare N (1)-alkyl-3 '-method of oximido derivatives of indirubin (I), wherein the anhydrous polar solvent that uses in the step (1) is tetrahydrofuran (THF) or N, dinethylformamide or N,N-dimethylacetamide; Described cooling temperature is between-10～5 ℃; Added basic-type accelerator is for being selected from Na ₂CO ₃, K ₂CO ₃, NaOH, KOH and NaH inorganic base substance and comprise the strong organic basic material of DBU, the mol ratio of itself and raw material Indirubin is 1.0～1.8.

9, preparation method as claimed in claim 8, wherein said anhydrous polar solvent is N, dinethylformamide or N,N-dimethylacetamide; Described basic-type accelerator is NaH and DBU, and the mol ratio of itself and raw material Indirubin is 1.3～1.6, and reinforced and temperature of reaction are-2～5 ℃.

10, preparation N as claimed in claim 1 (1)-alkyl-3 '-oximido derivatives of indirubin (I) method, the halohydrocarbon RX that uses in the step (1) wherein, described R group is selected from C1～5 alkyl, C2～5 alkene and aryl, described X group is Br or I, and the mol ratio of RX and starting raw material Indirubin is 1.0～1.8.

11, preparation method as claimed in claim 10 wherein when R is alkyl, is straight chain, branched and corresponding cyclic alkyl; When R is alkene, be the monoolefine of straight chain or band branched-chain alkene; When R is aryl, for benzyl, have the phenyl of single or multiple electron-withdrawing groups; The mol ratio of RX and starting raw material Indirubin is 1.3～1.6.

12, N as claimed in claim 1 (1)-alkyl-3 '-oximido derivatives of indirubin (I) preparation method, wherein Indirubin is dissolved in N, in the dinethylformamide (DMF), under-2 ~ 5 ℃ of conditions, add NaH, making the mol ratio of itself and raw material Indirubin is 1.3 ~ 1.5; Then, add halohydrocarbon RX, the mol ratio of RX and starting raw material Indirubin is 1.3 ~ 1.5; Reaction finishes to be determined as foundation with TLC, after the end, with pouring in the frozen water under the reaction solution stirring, separates out intermediate II, and after filtration, washed several times with water gets crude product II, through purification by silica gel column chromatography, gets high N (the 1)-alkyl derivatives of indirubin (II) of purity; Intermediate II and azanol are to reflux in 25% ethanol in water content, make the Compound I crude product, through acetone recrystallization, N (1)-alkyl-3 '-oximido derivatives of indirubin (I).

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